Process for preparing purified nucleic acid and the use thereof

ABSTRACT

The invention relates to a nucleic acid preparation with a content of below 1% protein, preferably below 0.1% protein, free of ethidium bromide, phenol, cesium chloride and detergents based on octyl phenol poly(ethylene glycol ether)n and with a content of below 1 EU/mg DNA of endotoxins. Said preparation is suitable as a drug particularly in gene therapy.

[0001] The invention concerns the preparation of purified nucleic acidand its use especially in gene therapy.

[0002] Replicatable nucleic acid is usually produced by amplifyingreplicatable plasmid DNA in gram-negative bacteria such as e.g. E. coli.After lysis of the biomass (usually alkaline lysis with lysozyme orultrasound), it is centrifuged and the supernatant is shaken out withphenol subsequently an ultracentrifugation on a caesium chloridegradient is carried out (Birnboim & Doly, Nucleic Acid Res. 7 (1979)1513-1523, Garger et al., Biochem. Biophys. Res. Comm. 117 (1983)835-842). However, such preparations contain endotoxins, phenol, caesiumchloride and/or ethidium bromide as a dye.

[0003] A further process is described in the QIAGEN® Plasmid Handbook(Qiagen Inc., Chatsworth, USA) and EP-B 0 268 946. According to thisprocess the cell lysate obtained after a conventional lysis ischromatographed on QIAGEN® -TIP, which contains QIAGEN® resin (a supportmaterial based on silica). The disadvantage of this process is that DNAbinding proteins are not completely detached from the DNA and thereforethe purified plasmid fraction contains proteins and in particularendotoxins (from the membrane of the gram-negative host cells) inconsiderable amounts.

[0004] In another process after alkaline lysis of the E. coli biomassthe centrifugation supernatant is chromatographed according to Birnboim& Doly under high salt conditions over anion exchange columns (e.g.Mono-Q, Source-Q from Pharmacia, Macroprep-Q from BioRad, Poros-Q fromPerseptive Biosystems or HyperD-Q from Biosepra, cf. Chandra et al.,Analyt. Biochem. 203 (1992) 169-172; Dion et al., J. Chrom. 535 (1990)127-147). Also in this case the purified plasmid fraction containsproteins and in particular endotoxins in considerable amounts.

[0005] In another process after alkaline lysis and subsequentphenol/chloroform extraction it is possible to chromatograph by gelfiltration (McClung & Gonzales, Analyt. Biochem. 177 (1989) 378-382;Raymond et al., Analyt. Biochem. 173 (1988) 125-133). Even after thispurification the plasmid preparation contains impurities and inparticular phenol.

[0006] A process for the isolation and purification of nucleic acids foruse in gene therapy is described in WO 95/21177 in which thepurification is essentially carried out by centrifugation, filtration,affinity chromatography or chromatography on an inorganicchromatographic material with subsequent chromatography on an ionexchanger. An additional removal of endotoxins can then be achievedaccording to WO 95/21177 when the nucleic acid is treated with anendotoxin removal buffer which contains 10% Triton®X100 and 40 mmol/lMOPS buffer (3-morpholino-1-propanesulfonate buffer). A disadvantage ofthis process is that the nucleic acid purified in this manner containsimpurities of Triton® and MOPS buffer. Although endotoxins can beremoved by this process to a content of ca. 100 EU/mg DNA (Qiagen News1/96, 3-5), it is not possible to remove endotoxins to a greater extentby this process.

[0007] However, for a therapeutic application such as for example forgene therapy a nucleic acid preparation is required which is as free aspossible of all impurities (in particular substantially free ofendotoxins). Above all the endotoxin content of plasmid preparations hasbeen hitherto an unsolved problem as described for example by Cotten etal., Gene Therapy 1 (1994) 239-246. A reduced endotoxin content (ca. 100EU/mg DNA) can only be achieved by the state of the art such as forexample according to WO 95/21177 if the nucleic acids are treated withnon-ionic detergents such as e.g. Triton (endotoxin removal buffer fromWO 95/21177). However, Triton® has a biological action such as e.g. lungchanges or reduction of blood pressure (Fiedler, “Lexikon der Hilfstoffefür Pharmazie und Kosmetik und angrenzende Gebiete (Band 9, 3rd edition,1989, Editio Cantor, DE)). The MOPS buffer which is additionallyrequired also contains a substance that is problematic with regard to atherapeutic application.

[0008] The invention provides a nucleic acid preparation, preferably aplasmid DNA, of high purity in which endotoxins are substantiallyremoved and preferably without ethidium bromide, phenol, caesiumchloride, polymyxin or non-ionic detergents and also provides a simpleand effective process for purifying such nucleic acids in particular forremoving endotoxins.

[0009] The invention concerns a nucleic acid that can be replicated ingram-negative bacteria, preferably a plasmid DNA with a content of lessthan 1% protein, preferably less than 0.1% protein and a content of lessthan 1 EU/mg DNA, preferably 0.01-0.1 EU/mg DNA of endotoxins. Thisplasmid DNA is preferably free of ethidium bromide, phenol and caesiumchloride, free of detergents based on octylphenolpoly(ethylene glycolether)n such as Triton® detergents and also free of MOPS buffersubstance and RNAse.

[0010] Amplification is understood as an increase in the copy number ofa nucleic acid (in particular DNA and plasmid DNA) based on thereplication of a vector. In this process numerous copies are producedfrom a template. A vector is replicated which represents the nucleicacid or which contains the cloned nucleic acid.

[0011] A plasmid DNA is understood as an extrachromosomal DNA duplexmolecule. The size of a DNA plasmid is usually 1 to more than 200 kb andone to several hundred copies are present in host cells. Plasmid DNA isusually amplified in gram-negative bacteria such as e.g. E. coli andsubsequently isolated. Plasmids are often used to construct cloningvectors, for the transfection of prokaryotic and eukaryotic cells. Atherapeutic use is of especial importance in connection with in vivo andex vivo gene therapy. Plasmid DNA that is used therapeuticallypreferably has a length of 5 to 20 kb, particularly preferably 5-10 kband is double-stranded. The plasmid DNA can be linearized or circularlyclosed. Preferably DNA is used that is essentially circularly closed.

[0012] Consequently the invention additionally concerns a pharmaceuticalcomposition containing a nucleic acid according to the invention,preferably plasmid DNA, in a therapeutically effective amount andoptionally additional pharmaceutical auxiliary substances, fillers oradditives.

[0013] Endotoxins are lipopolysaccharides from gram-negative bacteria.Endotoxins can have a pyrogenic effect in mammals and induce anendotoxin shock. The main toxic component of endotoxins is lipid A, thepolysaccharide moiety mediating the water solubility and the lipidmoiety having the toxic effect. The biological effect of endotoxins inmammals are in particular a hypersensitization as well as otherreactions which are accompanied by fever.

[0014] Plasmid DNA is amplified by standard methods in E. coli i.e. agram-negative bacterium. After fermentation the biomass obtained in thisprocess is lysed and the cells are lysed. In this process the endotoxinsare released from the cell membrane. This means that after amplificationof nucleic acids, in particular of plasmid DNA, in gram-negativebacteria and in particular in E. coli it is necessary to removeendotoxins if it is intended to use this plasmid DNA therapeutically.

[0015] Depending on the application doses of 50 μg to 10 mg and more areused or planned for a therapeutic application of replicatable nucleicacids, in particular of plasmid DNA. The dose amount depends on thedisease and type of administration. In an aerosol, e.g. for thetreatment of cystic fibrosis, doses of 400 μg and more are used. Thisapplies likewise to plasmid DNA encapsulated in a lipid complex (e.g. inliposomes). In order to provide such amounts of replicatable nucleicacid that can be used therapeutically, it is necessary to produce thereplicatable nucleic acid on a large scale. For this fermentationpreparations are expedient with 1-5 kg biomass from which 1-5 g nucleicacid can be isolated.

[0016] The invention also concerns a process for the production of aplasmid DNA with a content of less than 1 EU/mg DNA, preferably 0.01-0.1EU/mg DNA of endotoxins which is characterized in that plasmid DNA isreplicated in gram-negative bacteria, the biomass is lysed and thesoluble components are chromatographed on hydroxylapatite andsubsequently the said plasmid DNA is isolated. Before chromatography onhydroxylapatite it is preferably to carry out an ion exchangechromatography which essentially removes RNA and foreign proteins. Thiscan optionally remove further impurities and achieve a content ofnucleic acid of less than 1% protein, preferably less than 0.1% protein,free of ethidium bromide, phenol and caesium chloride. Such apreparation is also preferably free of detergents based on octylphenolpoly(ethylene glycol ether)n and MOPS buffer substance.

[0017] The process according to the invention enables numerousimpurities to be avoided or removed which plasmid DNA contains if it isproduced by a process familiar to a person skilled in the art. Above allit is surprisingly possible to drastically reduce the endotoxin contentin a simple manner.

[0018] In the process according to the invention an outstanding removalof endotoxins is achieved by the chromatography with hydroxylapatite.This is all the more surprising since chromatography on hydroxylapatiteis only used in the literature to separate DNA and RNA (Johnson & Ilan,Analyt. Biochem. 132 (1983) 20-25).

[0019] The chromatographic effect of hydroxylapatite is essentiallybased on the interaction between calcium²⁺ groups and the negativecharge of the nucleic acid to be purified and to a lesser extent on theinteraction of the nucleic acid to be purified with PO₄ ³⁻ groups on thesurface of crystalline hydroxylapatite (cf e.g. Protein PurificationMethods, Ed. by Elv. Harries and S. Angal, Oxford University Press 1989,238-244). Chromatography on hydroxylapatite can be essentially referredto as an ion exchanger step for nucleic acids in which the bound DNAcannot be eluted from the hydroxylapatite matrix by a simple-increase ofionic strength (e.g. NaCl) but rather by increasing the concentration,preferably of phosphate or citrate, divalent metal ions and/or EDTA.

[0020] In the process according to the invention endotoxins and thenucleic acid to be purified are firstly bound to hydroxylapatiteprimarily via dipole-dipole interactions in the chromatography onhydroxylapatite (e.g. HA-ceramic, BioGel HPHT, Bio-Gel HT/HTP fromBiorad DE, HA-Ultrogel from IBF or HA spheroidal from BDH, Macrosorb Cfrom Sterling Organics). The equilibration is usually carried out atneutral pH in phosphate buffer. A denaturing agent is preferably added,as in the subsequent washing of the column. Surprisingly it is possibleto displace the nucleic acid from its binding to hydroxylapatite withphosphate, citrate or calcium ions whereas the endotoxins remain bound.Instead of calcium, the displacement of the nucleic acid from itsbinding to hydroxylapatite can be achieved with other divalent metalions which can replace calcium in the apatite such as e.g. Mg, Fe, Mn.For the elution the ion concentration is preferably 100 mmol/l or more.Ion concentrations between 100 and 500 mmol/l or 200 and 500 mmol/l areparticularly preferred. A solution containing phosphate ions (e.g.phosphate buffer) is particularly preferably used. Before the elution(without denaturing agent) it is expedient to wash (with denaturingagent). It has turned out to be advantageous to for example use aphosphate of sulfate solution (100-200 mmol/l) to which a denaturingagent (e.g. urea or guanidine hydrochloride) has been added at aDNA-denaturing concentration (e.g. 6 mol/l urea).

[0021] In a preferred embodiment an ultrafiltration is additionallycarried out after the chromatography on hydroxylapatite.

[0022] In order to produce the nucleic acid according to the inventionthe plasmids which represent or contain the nucleic acid are usuallyamplified in gram-negative bacterial cultures. Such methods are known toa person skilled in the art and are described for example by Sambrook etal. (1989), Molecular Cloning: A Laboratory Manual, 2nd edition, ColdSpring Harbor Laboratory Press and by F.M. Ausubel et al., eds. (1991),Current Protocols in Molecular Biology, Wiley Interscience, New York.For this the bacterial cultures which contain the plasmids are firstlysubcultured and subsequently cultured in a suitable medium optionallywith addition of a selection agent.

[0023] The biomass is also lysed by methods familiar to a person skilledin the art (mechanical or enzymatic lysis, see e.g. Birnboim and Doly,Nucleic Acids Research 7 (1979) 1513-1423) without addition of RNAse. Itis possible to omit shaking out with phenol if the proteins areseparated by chromatography on an anion exchanger. After lysis andseparation of the insoluble components, preferably by centrifugation andfiltration over a filter candle (5 μm pores), the cell supernatant ispreferably firstly chromatographed on an anion exchanger to removeproteins. Suitable anion exchangers are anion exchangers based onagarose such as for example Q-Sepharose. Other suitable anion exchangersare based on polymethacrylate (Macroprep/Bio-Rad, Germany),polystyrene/divinylbenzene (Poros/Perseptive, HyperD/Biosepra,Source/Pharmacia) or silica gel on the surface of whichdiethylaminoethyl (DEAE) or dimethylaminoethyl (DMAE) groups are forexample bound.

[0024] In order to optimize the purification effect, the nucleic acid iseluted by means of a high salt gradient e.g. NaCl gradient (preferably0.65 mol/l-0.85 mol/l) in TE buffer. This surprisingly enables numerousimpurities (RNA, protein) to be separated in one step.

[0025] It is also preferable to carry out an additionalisopropanol/ethanol precipitation, preferably after the hydroxylapatitechromatography, to minimize the bioburden and for desalting.Subsequently the nucleic acid according to the invention can be bottledunder sterile. conditions.

[0026] The following examples describe the invention in more detail.

EXAMPLE 1 Isolation of Nucleic Acid from E. coli Biomass

[0027]E. coli biomass containing plasmid DNA is lysed by an alkalinelysis and the released plasmid DNA is chromatbgraphed over Q-Sepharoseand HA-Ceramic. The eluate is desalted by an isopropanol/ethanolprecipitation and concentrated and the plasmid DNA precipitate isresuspended in TE buffer.

[0028] Resuspension buffer: 50 mmol/l Tris/HCl, 10 mmol/EDTA-Na₂, pH8.0±0.2

[0029] Potassium acetate buffer: 3 mol/l potassium acetate buffer pH 5.5

[0030] 60 g biomass (wet, E. coli) from the fermenter is filled intodepyrogenized centrifuge beakers. 750 ml resuspension buffer is addedand it is stirred slowly (ca. 35 rpm) for at least 24 hours at 5±4° C.until the biomass is completely suspended. During this process thetemperature of the suspension is slowly increased to 25° C.

[0031] 750 ml 0.2 mol/l NaOH/1% SDS is added to the suspension whilestirring at ca 80 rpm and incubated for 5 minutes at 25° C. 750 mlpotassium acetate buffer (see above) is added while stirring and thetemperature of the biomass is lowered as rapidly as possible to 4° C.

[0032] The biomass is centrifuged for 30 minutes at 26000×g and 4° C.The supernatant which contains the plasmid DNA is decanted and filteredclear over a 5 μm filter candle.

[0033] Chromatography on Q-Sepharose ff:

[0034] TE buffer: 10 mmol/l Tris-HCl, 1 mmol/l EDTA pH 8.0±0.2

[0035] Equilibration/wash buffer=gradient buffer A: 10 mmol/l Tris-HCl,1 mmol/l EDTA, 0.65 mol/l NaCl pH 8.0±2.

[0036] Gradient buffer B: 10 mmol/l Tris-HCl, 1 mmol/l EDTA, 0.85 mol/lNaCl pH 8.0±0.2.

[0037] The decanted centrifuge supernatant is adjusted to 49-50 mS/cmconductivity by addition of ca. 350 ml TE buffer/l centrifugationsupernatant and cooled to 5°±4° C. The whole chromatography is carriedout at this temperature. The centrifugation supernatant is applied tothe equilibrated column at 5-8 column volumes (CV)/h. Subsequently thecolumn is washed at a flow rate of 5-8 CV/h with ca. 8 CV 10 mmol/lTris-HCl, 1 mmol/l EDTA, 0.65 mol/l NaCl pH 8.0±0.2.

[0038] Elution

[0039] A gradient is applied to the column (5 CV buffer A, 5 CV bufferB) and the eluate is fractionated at a flow rate of 5-8 CV/h. Thedetection is carried out at 254 nm. The pre-peak (impurities) isseparated from the main peak (plasmid DNA) by collecting the main peakfrom the increasing flank onwards in a separate vessel. The endotoxincontent of the eluate is between 1200 and 12000 EU/mg plasmid DNA.

[0040] Chromatography on Hydroxylapatite (HA Ceramic)

[0041] The chromatography is carried out at 5±4° C.

[0042] Equilibration buffer: 0.1 mol/l potassium phosphate, 6 mol/l ureapH 7.0±0.2

[0043] wash buffer 1: 0.15 mol/l potassium phosphate, 6 mol/l urea pH7.0±0.2

[0044] wash buffer 2: 0.02 mol/l potassium phosphate buffer pH 7.0±0.2

[0045] elution buffer: 0.5 mol/l potassium phosphate pH 7.0±0.2

[0046] The detection is carried out at 254 nm with a UV detector/plotterunit. A 1% product solution (plasmid DNA) measured with a calibratedphotometer is used as a calibration solution.

[0047] The Q-Sepharose® pool is adjusted to a final concentration of 1.1mmol/l calcium chloride and applied to the equilibrated column at a flowrate of 5-8 CV/h.

[0048] Subsequently the column is consecutively washed at a flow rate of5-8 CV/h with:

[0049] 1. 0.1 mol/l potassium phosphate, 6 mol/l urea pH 7.0±0.2, untilabsorbance is no longer detectable on the detector.

[0050] 2. 2-4 CV, 0.15 mol/l potassium phosphate, 6 mol/l urea pH7.0±0.2

[0051] 3. 5 CV, 0.02 mol/l potassium phosphate pH 7.0±0.2

[0052] Following the wash steps it is eluted with 0.5 mol/l potassiumphosphate buffer pH 7.0±0.1 at a flow rate of 5-6 CV/h.

[0053] The peak is collected, heated to 25° C. and 10% of it's volume of4 mol/l KCl solution is added. Subsequently 0.7 parts by volume(relative to the volume of the eluate) of isopropanol is added, thesolutions are mixed and incubated for 5-10 minutes at 25° C. It iscentrifuged for 30 minutes at ≧20,000×g, the plasmid DNA being in theprecipitate.

[0054] 20 ml 70% ethanol is added to the precipitate and it is againcentrifuged for 10-15 minutes at ≧20,000×g at 4° C.

[0055] The precipitate which contains the plasmid DNA is resuspended inTE buffer (10 mmol/l Tris-HCl, 1 mmol/l EDTA pH 8.0+0.2) and adjusted toa plasmid concentration of 1 mg/ml. The endotoxin content is typicallyless than 0.06 EU/mg DNA and between 0.01 and 0.06 EU/mg DNA.

[0056] The endotoxin content is determined by adding a limulusamoebocyte lysate solution (LAL solution) to the solution to beexamined. Endotoxins result in a gel formation in this mixture.

[0057] No gel formation should occur in the negative controlpreparations and in the positive control preparations as well as in thesample solutions supplemented with two λ control standard endotoxin agel formation must occur.

[0058] The first dilution step of the solution of active substance forwhich these criteria apply and in which no gel formation occurs is usedto calculate the endotoxin content of the solution of active substancesolution according to the following formula:

E=V×λ(EE/ml)

[0059] E: endotoxin content

[0060] V: dilution factor

[0061] λ: lysate sensitivity (EE/ml)

EXAMPLE 2

[0062] Plasmid Preparation According to the State of the Art

[0063] The plasmid preparation is carried out analogously to Birnboim etal., Nucl. Acids Res. 7 (1979) 1513-1523 and Meth. Enzymol. 100 (1983)243-255. Accordingly the bacterial cells are lysed in NaOH/SDS in thepresence of RNase. It is centrifuged and the supernatant which containsthe plasmid DNA is processed further. The supernatant is loaded onto apre-equilibrated Qiagen® column.

[0064] The bacterial mass is resuspended in 4 ml buffer (100 μg/ml RNaseA, 50 mmol/l Tris-HCl, 10 mmol/l EDTA, pH 8.0). 4 ml lysis buffer (200mmol/l NaOH, 1% SDS) is added and it is incubated for 5 minutes at roomtemperature. Subsequently 4 ml neutralisation buffer (3 mol/l potassiumacetate, pH 5.5) is added and it is incubated for 15 minutes at 4° C. Itis centrifuged for 30 minutes at 30,000×g at the same temperature andthe supernatant is processed further. A Qiagen® column is equilibratedwith 4 ml equilibration buffer (750 mmol/l NaCl, 50 mmol/l MOPS, 15%ethanol, pH 7.0, 0.15% Triton®X 100) and the supernatant is applied tothe column. It is washed with 1 mol/l NaCl, 50 mmol/l MOPS, 15% ethanol,pH 7.0 and eluted with 5 ml elution buffer (1.25 mol/l NaCl, 50 mmol/lTris-HCl, 15% ethanol, pH 8.5).

[0065] The eluate is precipitated with isopropanol (0.7 vol) andcentrifuged for 30 minutes at 15,000×g at 4° C. The DNA pellet is washedin 70% ethanol and again centrifuged. Subsequently the pellet isresolubilized in 10 mmol/l Tris-HCl, 1 mmol/l EDTA, pH 8.0.

[0066] The endotoxin content of such a plasmid preparation is typically300-3000 EU/mg. Using an endotoxin removal buffer according to WO95/21177 and Qiagen news 1/96, p. 3-5 the endotoxin content can befurther reduced to ca. 100 EU/mg.

[0067] List of References

[0068] Ausubel, F. M., et al. eds. (1991), Current Protocols inMolecular Biology, Wiley Interscience, New York

[0069] Birnboim, H. C. and Doly, J., Nucleic Acids Research 7 (1979)1513-1523

[0070] Birnboim, H. C., et al., Meth. Enzymol. 100 (1983) 243-255

[0071] Chandra et al., Analyt. Biochem. 203 (1992) 169-172

[0072] Cotten et al., Gene Therapy 1 (1994) 239-246

[0073] Dion et al., J. Chrom. 535 (1990) 127-147

[0074] European Patent EP-B 0 268 946

[0075] Fiedler, Lexikon der Hilfsstoffe für Pharmazie und Kosmetik undangrenzende Gebiete (Vol. 9, 3rd edition, 1989, Editio Cantor, GER)

[0076] Garger et al., Biochem. Biophys. Res. Comm. 117 (1983) 835-842

[0077] Johnson & Ilan, Analyt. Biochem. 132 (1983) 20-25

[0078] McClung & Gonzales, Analyt. Biochem. 177 (1989) 378-382

[0079] Protein Purification Methods, Ed. by Elv. Harries and S. Angal,Oxford University Press 1989, 238-244

[0080] QIAGEN NEWS 1/96, 3-5

[0081] QIAGEN Plasmid Handbook (Qiagen Inc., Chatsworth, USA)

[0082] Raymond et al., Analyt. Biochem. 172 (1988) 125-133

[0083] Sambrook, J. et al. (1989), Molecular Cloning: A LaboratoryManual, 2nd edition, Cold Spring Harbor Laboratory Press, New York, USA

[0084] WO 95/21177

1-10. (Canceled)
 11. DNA preparation comprising a DNA, proteins in anamount of less than about 0.1% DNA and endotoxins in an amount of lessthan about 1 EU/mg DNA.
 12. The DNA preparation of claim 11, comprisingendotoxins of less than about 0.06 EU/mg DNA.
 13. The DNA preparation ofclaim 11, comprising endotoxins of about 0.01 to 0.1 EU/mg DNA.
 14. TheDNA preparation of claim 11, wherein the DNA preparation is free ofethidium bromide, phenol, cesium chloride, octylphenolpoly(ethyleneglycol ether)n detergents and MOPS buffer.
 15. The DNA preparation ofclaim 11, wherein the DNA can be replicated in gram-negative bacteria.16. The DNA preparation of claim 15, wherein the gram-negative bacteriais Escherichia coli.
 17. The DNA preparation of claim 11, wherein theDNA is plasmid DNA.
 18. The DNA preparation of claim 17, wherein theplasmid DNA is capable of replication.
 19. A pharmaceutical compositionsuitable for gene therapy comprising a therapeutically effective amountof the DNA preparation of claim 15 and a pharmaceutically acceptablecarrier.
 20. The composition of claim 19, wherein the DNA preparationcontains endotoxins in an amount of less than about 0.06 EU/mg DNA. 21.The composition of claim 19, wherein the DNA preparation containsendotoxins in an amount of about 0.01 to 0.1 EU/mg DNA.
 22. Thecomposition of claim 19, wherein the DNA is a plasmid DNA.
 23. Thecomposition of claim 22, wherein the plasmid DNA is encapsulated inliposomes.
 24. A method for gene therapy of cystic fibrosis caused bythe absence of a normal first gene or the presence of a defective secondgene, comprising administering an effective amount of the DNApreparation of claim 18 to a patient in need thereof, wherein theplasmid DNA contains said normal first gene or a normal second genecorresponding to the defective second gene.
 25. A process for making theDNA preparation of claim 11, comprising the following steps (a)providing gram-negative bacteria containing said DNA; (b) lysing saidbacteria to obtain a lysate, wherein the lysate is a DNA-containingfraction and thereafter (c) chromatographing said DNA-containingfraction on hydroxylapatite in order to obtain said DNA preparation. 26.The method of claim 25, further comprising replicating said DNA ingram-negative bacteria after step (a) and before step (b).
 27. Themethod of claim 26, further comprising transfecting the gram-negativebacteria with a cloning vector containing said DNA before step (a). 28.The method of claim 26, further comprising eluting said hydroxylapatitein step (c) with a solution of phosphate, citrate, sulfate or divalentmetal ions to obtain said DNA preparation.
 29. The method of claim 25,further comprising, after step (b) and before step (c), filtering thelysate to obtain a filtrate and fractionating said filtrate by gelfiltration to obtain said DNA-containing fraction.
 30. The method ofclaim 26, further comprising, after step (b) and before step (c),filtering the lysate to obtain a filtrate and fractionating saidfiltrate by gel filtration to obtain said DNA-containing fraction. 31.The method of claim 27, further comprising, after step (b) and beforestep (c), filtering the lysate to obtain a filtrate and fractionatingsaid filtrate by gel filtration to obtain said DNA-containing fraction.32. The method of claim 28, further comprising, after step (b) andbefore step (c), filtering the lysate to obtain a filtrate andfractionating said filtrate by gel filtration to obtain saidDNA-containing fraction.